This invention relates to treating oxidative stress and, particularly, to treating disease conditions associated with a presence of superoxide and of reactive chemical species derived from superoxide.
The oxygen radicals (including for example xe2x80x9csuperoxidexe2x80x9d, O2xc2x7xe2x88x92; hydroxyl radical, OHxc2x7) and some non-radical derivatives of O2 (including for example hydrogen peroxide, H2O2) are commonly known as reactive oxygen species [xe2x80x9cROSxe2x80x9d]. During electron transport in redox reactions in all living organisms the reduction of molecular oxygen by a single electron generates the reactive oxy-radical, i.e., the superoxide anion (O2xc2x7xe2x88x92). ROS, including superoxide and chemical species derived from it, can be damaging to living systems. In healthy aerobic organisms, production of ROS (and of reactive nitrogen species [xe2x80x9cRNSxe2x80x9d]) is balanced by antioxidant defenses. The balance is imperfect, however, and some ROS- and RNS-mediated damage is ongoing to greater or lesser degrees. In healthy organisms, damaged biomolecules are continually repaired or replaced.
A serious imbalance that arises between production of ROS/RNS and the antioxidant defenses is referred to as xe2x80x9coxidative stressxe2x80x9d, and significant oxidative damage can be caused by oxidative stress. Oxidative stress can result from diminished antioxidants (for example by depletion or underproduction of antioxidants as a result of dietary insufficiency or genetically associated metabolic deficiency), or from increased production of ROS/RNS (for example by exposure to elevated O2 or by metabolism of toxins or by excessive activation of ROS/RNS-producing pathways resulting from inflammatory disease processes).
Oxidative stress is associated with a wide variety of disease conditions. In some instances oxidative stress is a primary cause of the disease condition (e.g., radiation-induced damage, some cancers, some drug side effects, and sometimes atherosclerosis and hypertension); or probably is a primary cause (e.g., Vitamin E deficiency and Selenium deficiency). In other instances oxidative stress is secondary, but may contribute significantly to pathology (e.g., atherosclerosis, rheumatoid arthritis, and inflammatory bowel disease, and possibly a significant number of other diseases).
Under normal circumstances in cytosol or on extracellular surfaces the superoxide radical is consumed by superoxide dismutases (SODs). SOD enzymes are oxoreductases (EC 1.1.5.1.1) that contain copper, iron, or manganese ion in the active site and catalyze the dismutation of O2xc2x7xe2x88x92 radicals to molecular oxygen and hydrogen peroxide, which is further converted to molecular oxygen and water by catalase. Many mammalian diseases may be characterized as conditions in which the body fails to contain an overproduction of the O2xc2x7xe2x88x92 radical and of the more harmfully reactive Oxc2x7 . radical which is consequently derived through Fenton chemistry between reduced metal ions (Cu+, Fe2+) and hydrogen peroxide.
In certain metabolic processes, the production of O2xc2x7xe2x88x92 is enhanced, resulting in tissue injury and disease. Examples of such oxidative stress-related diseases include perfusion injury, such as that which occurs after acute myocardial infarction or stroke, inflammatory processes such as arthritis, inflammatory bowel conditions and stomach ulcers.
Considerable effort has been directed toward developing pharmaceuticals that might be therapeutically useful in ameliorating tissue injury and disease associated with oxidative stress. In one general approach, an excess of reactive oxygen species would be corrected by administering an agent that would produce increased superoxide dismutase activity where oxidative stress is either underway or likely to occur. Attempts have been made, for example, to administer a superoxide dismutase (typically, a CuZn-SOD) enzyme of animal origin to a subject in need of treatment. The SOD may be used in the native state, but some have proposed modifying it in some manner, such as by treatment with albumin (see, e.g., L. G. Cleland et al. 1979, Arthritis Rheum., Vol. 22, p. 559) polyethylene glycol (see, e.g., J. M. McCord et al. 1979, in, xe2x80x9cExcerpta Medicaxe2x80x9d, Ciba Foundation Symposium, Vol. 65, pp. 343-60), ficoll (see, e.g., W. F. Petrone et al. 1980, Proc. Nat""l. Acad. Sci. USA, Vol. 77, pp. 1159-63), polyalkylene glycol (see, e.g., Japanese Patent No. 61-249388 (Ajinomoto Co., 1985)) or liposome (see, e.g., A. M. Michelson 1982, Agents Action, Vol. 11, pp. 179-210). Modification of SOD enzymes may provide advantages for their use as drugs. A suitably modified SOD may, for example, have longer lifetime in vivo, or may have reduced toxicity as compared to the native enzyme, or may have reduced immunogenicity (see, e.g., A. Abuchowski et al. 1978, Recl. Trav. Chim. Pays-Bas, Vol. 97, pp. 293-95). However, such modified enzymes may be costly, and the modifications may reduce their effective catalytic activity.
Others have proposed administering synthetic lower molecular weight metal complexes, and a variety of small-molecule SOD xe2x80x9cmimicsxe2x80x9d have been developed and tested for pharmaceutical efficacy (see, e.g., B. Halliwell et al. 1999, Free Radicals in Biology and Medicine, 3d Ed., Oxford, see particularly, pp. 831-32). Where such SOD mimics employ copper or iron as the metal, however, they may generate highly reactive OHM radicals, which can rapidly interact with surrounding living materials, resulting in tissue injury and aggravation of the disease state.
In one general aspect the invention features a dendrimer construct having a core and two or more branched arms projecting outwardly from the core. The arms include internal branched units and terminal moieties; the terminal moieties constitute an outermost surface of the dendrimer construct. The arms include at least one metal ion binding site enclosed within the outermost surface. The outermost surface of the dendrimer construct is sufficiently densely packed to restrict the movement of larger molecules from the milieu into the dendritic construct, and the surface is sufficiently porous to permit free movement of smaller molecules from the milieu into the dendrimer construct and to the metal ion binding site and out from the dendrimer construct to the milieu.
In another general aspect the invention features a metal-dendrimer complex in which a metal active site is enclosed within the surface of the dendrimer construct. The dendrimer construct includes a core and two or more branched arms projecting outwardly from the core. The arms include internal branched units and terminal moieties; the terminal moieties constitute the outer surface of the dendrimer construct. The arms include at least one metal ion binding site associated with one or more internal branched units and enclosed within the outermost surface, and a metal ion is complexed at the metal ion binding site to form the metal active site. The outermost surface of the dendrimer construct is sufficiently densely packed to restrict the movement of larger molecules from the milieu into the dendritic construct Smaller molecules such as the superoxide anion (O2xc2x7xe2x88x92) move freely from the milieu into the dendrimer construct and to the metal active site, where the dismutation of superoxide to hydrogen peroxide is effected; and smaller molecules such as hydrogen peroxide move freely out from the dendrimer construct to the milieu.
In some embodiments the metal ion in the complex is an ion of a transition metal such as copper, manganese, or iron; in particular embodiments the metal ion is an ion of copper, particularly copper(II); or is an ion of iron, particularly iron(III).
In some embodiments the dendrimer construct results from sequential monomer addition in a divergent synthesis, beginning from a core and constructing the branched arms by proceeding outwardly through successive generations (the core being the zerosth generation). One mode of such divergent synthesis proceeds by sequential addition of monomers using a protection-deprotection scheme. The core may have two (divalent), three (trivalent), four (tetravalent) or more reactive moieties, providing points of attachment for, respectively, two, three, four or more branched arms; usually the core is divalent or trivalent.
A The monomers or branching units making up the arms may be 1xe2x86x922 branching or 1xe2x86x923 branching. The dendrimer construct may have all structurally similar arms, and each arm may contain similar repeat internal branch units (except for one or more branch units with which the ion binding site is associated); or each arm may contain dissimilar internal branch units. Or, the dendrimer construct may have structurally different arms, each having either similar or dissimilar internal branch units or terminal moieties.
Preferred internal branch units, at least in the vicinity of metal active sites, include structures that are less likely to be degraded by the presence of the superoxide anion (O2xc2x7xe2x88x92) or of its derivatives such as the highly reactive OHxc2x7 radical which may exist transiently near the metal reactive site, or hydrogen peroxide.
In some embodiments the dendrimer construct is a dendritic polypeptide or a dendritic polyamidoamine. Suitable monomers include any of various L-, D-, or DL-xcex1-amino acids carrying functional groups in the side chains R, where R has the general formula (CH2)nNH2, where n=1-7; or the general formula (CH2)nCOOH, where n=1-7.
In some embodiments the metal ion is complexed with imidazole groups provided by L-histidine within the dendrimer construct Particular such embodiments include dendrimer constructs having 1,4-diaminobutane [xe2x80x9cDABxe2x80x9d] as a core and having L-lysine [xe2x80x9cKxe2x80x9d] as a monomer for each generation except that generation in which L-histidine [xe2x80x9cHxe2x80x9d] is used to provide imidazole coordination sites for copper ions. In some such embodiments 16 histidine residues are provided and, accordingly, histidine is employed as the monomer at the fourth generation; these include the D6 construct (DAB)K2K4K8H16K16K32, the D7 construct (DAB)K2K4K8H16K16K32K64, and the D8 construct (DAB)K2K4K8H16K16K32K64K128. Where, as in these embodiments, 16 imidazole groups are provided, the number of copper(II) ions that may be complexed in each molecule may be in the range from as few as 1 to as many as 4, more usually from 1 to 3 or from 1 to 2. In some embodiments the N(1) of the imidazole group is alkylated with an iodoalkane having the general formula I(CH2)nH(n=1-8). (See, A. Noordam et al. 1978, Recl. Trav. Chim. Pays-Bas, Vol 97, pp. 293-95.)
In another general aspect the invention features a method for treating or preventing a disease condition associated with oxidative stress, by administering to a subject in need of treatment a metal-dendrimer complex according to the invention, in a form and by a route of administration suitable for bringing the complex to the site of the condition. A disease condition associated with oxidative stress, as that expression is used herein, is one in which oxidative stress is a primary cause or in which oxidative stress is secondary to the condition but contributes significantly to disease pathology.
In some embodiments the disease condition to be treated is an oxidative stress associated disease of the gastrointestinal tract and the metal-dendrimer complex is administered orally for topical treatment within the gastrointestinal lumem. As will be appreciated, movement of molecules across the mucosa can be influenced by molecular size as well as various other properties, and absorption can be effected differently in different regions of the digestive tract. See, e.g., McMartin et al. 1987, Jour. Pharm. Sci, Vol. 76, pages 535 ff.; Peters et al. 1987, Jour. Pharm. Si., Vol. 76, pages 857 ff. A preferred metal-dendrimer complex for topical treatment in the digestive tract has a higher molecular weight, usually in the range about 50 kd to about 100 kd or greater, so that absorption of the complex across the digestive tract mucosa, and clearance of the complex through the kidneys, will be limited. In some embodiments a metal-dendrimer complex for treatment in the digestive tract has a molecular weight in the range about 50 daltons to about 100 daltons.
The water-soluble metal-dendrimer complexes according to the invention can be useful in treatment of any of a variety of disease conditions associated with oxidative stress. For example, a Copper(II)-dendritic peptide complex having molecular size in the range greater than about 50 kd can also be administered rectally, or by intravenous, intraperitoneal, intramuscular, or subcutaneous injection, for treatment of damage associated with oxidative stress in the digestive tract.
The dendrimer construct will not be a substrate for enzymatic degradation Moieties in the outermost tiers, on the surface of the dendritic construct, can be modified to facilitate affinity of the complex to cell surfaces or tissues, and particular modifications can be used to provide affinity for selected types of cells or tissues. The surface of the dendritic construct can be modified, for example by use of agents such as polyethylene glycol (see, e.g., C. O. Beauchamp et al. 1978, Anal. Biochem., Vol. 131, pp. 25-33) to reduce inununogenicity of the complex; or, for example, by use of alkyl groups to facilitate affinity of the construct to cell surfaces or tissues or to facilitate permeability of the construct across membrane bilayers; or, for example, by conjugation to the dendrimer construct of selected cationic peptide fragments, which have a high aiffity for heparin-like proteoglycans in targeted cells or tissues (see, e.g., M. Inoue et al 1991, Jour. Biol. Chem., Vol. 266, pp. 16409-14), to promote accumulation of the complexes at one or more targeted disease sites. The molecular size of the dendrimer complex can be readily controlled
Interaction of proteins or protein fragments with the surface of the dendrimer construct is expected not to substantially alter or destroy the tertiary structure of metal active site. Even where a surface-modified metal-dendrimer complex according to the invention is employed, small molecules such as the O2xc2x7xe2x88x92 radical can diffuse relatively unimpeded to the active site, where they participate in the redox reaction. The metal active site is situated deeply within the dendrimer construct. The extremely reactive and harmful hydroxyl radical (OHxc2x7), which is an expected product of Fenton chemistry at the active site, is rapidly converted to hydrogen peroxide before it can leave the dendrimer complex to the surrounding milieu because the hydroxyl radical has a short life time (xcx9c70 ns; I. Saito et al. 1990, Chemistry of Active Oxygens, chapter 1, p. 4, M. Misono, ed., Chemical Review No.7, Chemical Society of Japan, Tokyo (in Japanese)) and the rate constant k for the dismutation reaction OHxc2x7+OHxc2x7xe2x86x92H2O2 is extremely high (k=5xc3x9710xe2x88x929 Mxe2x88x921Sxe2x88x921; B. Halliwell et al., supra, p. 57).
The invention will now be described in further detail, with reference to the drawings.